title: GroEL/ES buffering and compensatory mutations promote protein evolution by stabilizing folding intermediates.
abstract: Maintaining stability is a major constraint in protein evolution because most mutations are destabilizing. Buffering and/or compensatory mechanisms that counteract this progressive destabilization during functional adaptation are pivotal for protein evolution as well as protein engineering. However, the interplay of these two mechanisms during a full evolutionary trajectory has never been explored. Here, we unravel such dynamics during the laboratory evolution of a phosphotriesterase into an arylesterase. A controllable GroEL/ES chaperone co-expression system enabled us to vary the selection environment between buffering and compensatory, which smoothened the trajectory along the fitness landscape to achieve a >10(4) increase in arylesterase activity. Biophysical characterization revealed that, in contrast to prevalent models of protein stability and evolution, the variants' soluble cellular expression did not correlate with in vitro stability, and compensatory mutations were linked to a stabilization of folding intermediates. Thus, folding kinetics in the cell are a key feature of protein evolvability.
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, BC V6T 1Z4, Canada.
authors.forename: Kirsten T
authors.initials: KT
authors.lastname: Wyganowski
authors.forename: Miriam
authors.initials: M
authors.lastname: Kaltenbach
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Tokuriki
mesh_terms: D018835:Chaperonin 10 / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D018834:Chaperonin 60 / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D019020:Directed Molecular Evolution; D004789:Enzyme Activation / Q000235:genetics; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding / Q000235:genetics; D017510:Protein Folding; D055550:Protein Stability; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D012641:Selection, Genetic; D012995:Solubility
chemical_list: D018835:Chaperonin 10; D018834:Chaperonin 60
keywords: 2-naphthyl hexanoate; 2NH; 4,4′-bis(1-anilinonaphthalene 8-sulfonate); GdnHCl; PTE; amp; ampicillin; bis-ANS; cam; chaperonins; chloramphenicol; guanidine hydrochloride; phosphotriesterase; protein engineering; protein evolution; protein stability; soluble functional expression